Dual Metronomic Chemotherapy with Nab-Paclitaxel and Topotecan Has Potent Antiangiogenic Activity in Ovarian Cancer Rebecca A

Published OnlineFirst October 29, 2015; DOI: 10.1158/1535-7163.MCT-14-0630

Small Molecule Therapeutics Molecular Cancer Therapeutics Dual Metronomic Chemotherapy with Nab-Paclitaxel and Topotecan Has Potent Antiangiogenic Activity in Ovarian Cancer Rebecca A. Previs1, Guillermo N. Armaiz-Pena1, Yvonne G. Lin2, Ashley N. Davis1, Sunila Pradeep1, Heather J. Dalton1, Jean M. Hansen1, William M. Merritt1, Alpa M. Nick1, Robert R. Langley3, Robert L. Coleman1, and Anil K. Sood1,3,4

Abstract

There is growing recognition of the important role of met- compared with vehicle (P < 0.01). In the HeyA8-MDR model, ronomic chemotherapy in cancer treatment. On the basis of metronomic monotherapy with either cytotoxic agent had their unique antiangiogenic effects, we tested the efficacy of modest effects on tumor growth, but combination therapy nab-paclitaxel, which stimulates thrombospondin-1, and topo- decreased tumor burden by 61% compared with vehicle tecan, which inhibits hypoxia-inducible factor 1-a,atmetro- (P < 0.03). The greatest reduction in MVD (P < 0.05) and nomic dosing for the treatment of ovarian carcinoma. In vitro proliferation was seen in combination metronomic therapy and in vivo SKOV3ip1, HeyA8, and HeyA8-MDR (taxane-resis- groups. Combination metronomic therapy resulted in pro- tant) orthotopic models were used to examine the effects of longed overall survival in vivo compared with other groups metronomic nab-paclitaxel and metronomic topotecan. We (P < 0.001). Tube formation was significantly inhibited in examined cell proliferation (Ki-67), apoptosis (cleaved cas- RF-24 endothelial cells exposed to media conditioned with pase-3), and angiogenesis (microvessel density, MVD) in metronomic nab-paclitaxel alone and media conditioned with tumors obtained at necropsy. In vivo therapy experiments combination metronomic nab-paclitaxel and metronomic topo- demonstrated treatment with metronomic nab-paclitaxel alone tecan. The combination of metronomic nab-paclitaxel and met- and in combination with metronomic topotecan resulted in ronomic topotecan offers a novel, highly effective therapeutic significant reductions in tumor weight (62% in the SKOV3ip1 approach for ovarian carcinoma that merits further clinical model, P < 0.01 and 96% in the HeyA8 model, P < 0.03) development. Mol Cancer Ther; 14(12); 1–10. 2015 AACR.

Introduction strengthened the rationale for the use of metronomic chemotherapy in many malignancies, including ovarian cancer (2–6). Angio- Metronomic dosing is deﬁned as the frequent administration of genic pathways and the tumor microvasculature are attractive a chemotherapeutic agent at low, minimally toxic doses. Metro- targets for cancer treatment, as supported by the robust number of nomic chemotherapy is believed to inhibit tumor growth primar- ongoing clinical trials in this arena (6, 7–13) in a variety of solid ily by targeting proliferating endothelial cells (1), with an expec- malignancies, including breast, prostate, neuroendocrine, hepa- tation of reduced side effects. Preclinical and clinical data have tocellular, colorectal, and ovarian cancer. The use of metronomic chemotherapy as a means of inhibiting angiogenesis is generating clinical interest (2, 5). 1Department of Gynecologic Oncology, The University of Texas MD Paclitaxel plays a critical role in frontline adjuvant therapy Anderson Cancer Center, Houston, Texas. 2Department of Obstetrics and Gynecology, Keck School of Medicine, University of Southern for ovarian cancer and has been shown to result in increased California, Los Angeles, California. 3Department of Cancer Biology, thrombospondin-1 (TSP-1) in the tumor microenvironment The University of Texas MD Anderson Cancer Center, Houston, Texas. (14). However, the cremophor diluent in paclitaxel may interfere 4 Center for RNA Interference and Non-Coding RNA, The University of with angiogenesis inhibition at metronomic doses (15, 16). Nab- Texas MD Anderson Cancer Center, Houston, Texas. paclitaxel is a cremophor-free compound that uses 130-nanome- Note: Supplementary data for this article are available at Molecular Cancer ter albumin-bound (nab) technology to circumvent the need for Therapeutics Online (http://mct.aacrjournals.org/). solvents. Nab-paclitaxel achieves a larger volume of distribution R.A. Previs, G.N. Armaiz-Pena, and Y.G. Lin contributed equally to this article. and greater suppression of tumor growth at metronomic doses Current address for G.N. Armaiz-Pena: Department of Basic Sciences, Division of than cremophor paclitaxel in many preclinical tumor models Pharmacology, Ponce Health Sciences University and Ponce Research Institute, (15, 17, 18). Nab-paclitaxel has demonstrated promising activity Ponce, Puerto Rico. Current address for W.M. Merritt: SC Oncology Associates, in patients with recurrent ovarian, fallopian tube, and primary Columbia, South Carolina. peritoneal cancers (19). Corresponding Author: Anil K. Sood, The University of Texas MD Anderson Camptothecins such as topotecan, which are known to inhibit Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-745- topoisomerase I, also have antiangiogenic properties (20–24). 5266; Fax: 713-792-7586; E-mail: [email protected] One known mechanism of action of topotecan is its ability to doi: 10.1158/1535-7163.MCT-14-0630 suppress expression of hypoxia-inducible factor (HIF)-1a, a key 2015 American Association for Cancer Research. hypoxia-induced transcription factor that regulates the expression

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of several proangiogenic genes such as VEGF (25, 26). Metro- Whole-cell lysates were prepared from both treated and untreated nomic topotecan has been found effective in multiple preclinical cells at 24-hour time points up to 96 hours using modified RIPA and clinical models (27–29), including ovarian cancer (20, 23, buffer. Forty micrograms of protein lysate was boiled for 5 30–34). We have previously shown that metronomic doses of minutes and then loaded onto a 8% polyacrylamide/SDS topotecan decrease cell proliferation and angiogenesis while gel, transferred to nitrocellulose, and incubated with anti-human simultaneously increasing apoptosis in murine ovarian cancer TSP-1 antibody overnight (1:100 dilution; Abcam) and carbonic models, effects likely attributable to reductions in VEGF and anhydrase IX (1:500; Novus Biologicals). Each Western blot HIF1a (20, 21). analysis was performed in triplicate. Equal loading was verified Several preclinical studies in solid malignancies such as breast using vinculin (1:3,000 dilution; Sigma-Aldrich). cancer and melanoma have previously evaluated the use of doublet combination metronomic therapy using metronomically Immunohistochemistry dosed cyclophosphamide and uracil-tegafur (a fluorouracil oral Immunostaining for Ki-67, cleaved caspase-3, CD31, and TSP-1 prodrug; ref. 35), metronomic low-dose metronomic vinblastine was performed on paraffin-embedded slides of ovarian cancer and low-dose metronomic oral cyclophosphamide (36), and low- tumors from HeyA8 and HeyA8-MDR orthotopic models to dose metronomic vinblastine and low-dose metronomic cyclo- determine the proliferative index of the cells (i.e., to determine phosphamide (37). The effectiveness of dual metronomic therapy the effects of treatment on tumor cell proliferation). Considering preclinically has prompted evaluation of combination metro- the proposed role of angiogenesis inhibition in the treatment of nomic chemotherapy in clinical trials (38–42). ovarian cancer, we also determined the microvessel density Given the unique and nonoverlapping mechanisms of action of (MVD). nab-paclitaxel and topotecan (i.e., increasing secretion of TSP-1 Slides were deparaffinized sequentially in xylene and declin- and inhibiting HIF1a), we investigated the effects of these agents ing grades of ethanol before rehydration. Antigen retrieval combined at metronomic doses in preclinical models of ovarian before Ki-67 and cleaved caspase-3 staining was performed by carcinoma. heating slides in a steam cooker for 10 minutes in 0.2 mol/L Tris buffer, pH 9.0, while antigen retrieval for CD31 and TSP-1 Materials and Methods immunohistochemical analysis was accomplished using proteinase K (DakoCytomation) at room temperature. Endog- Ovarian cancer cell lines enous peroxides were blocked with 3% H2O2 in methanol Experiments were conducted using taxane-sensitive (HeyA8 (Ki-67, cleaved caspase-3, and TSP-1) or PBS (CD31). Nonspe- and SKOV3ip1) and taxane-resistant (HeyA8-MDR) epithelial cific epitopes were blocked using 5% normal horse serum and ovarian cancer cell lines. Descriptions of the derivation and 1% normal goat serum at room temperature. Slides were then maintenance of these cell lines have been reported previously incubated with primary antibody to Ki-67 (1:200; DakoCyto- (43–45). In brief, HeyA8 and SKOV3ip1 cells were maintained in mation); cleaved caspase-3 rabbit polyclonal anti-human, RPMI-1640/15% FBS with 0.1% gentamicin sulfate (Gemini mouse, or rat (1:200; Biocare Medical); TSP-1 (1:100; Abcam); Bioproducts). HeyA8-MDR cells were grown in the same media or CD31 (1:800; PharMingen) at 4C overnight. After washing supplemented with 300 mg/mL paclitaxel. All experiments were the slides with PBS, we added the appropriate horseradish performed with cells at 70% to 80% confluence. The tissue- peroxidase–conjugated secondary antibody in blocking solu- specific microvascular endothelial cell line MOEC (murine ovar- tion for one hour at room temperature. Slides were developed ian endothelial cells) and immortalized human vascular endo- with 3,300-diaminobenzidine chromogen (Invitrogen) and thelial cells (RF-24) were used for the in vitro experiments; their counterstained with Gil No. 3 hematoxylin (Sigma-Aldrich). derivation and characterization have been previously reported The proliferative index was calculated by dividing the num- (46, 47). Cells were maintained in DMEM supplemented with ber of Ki-67–positive (brown) nuclei by the total number of pyruvate, amino acids, and penicillin/streptomycin. Cell lines cells for each of 5 randomly selected 200 high-power fields were obtained from the ATCC and routinely tested for absence per tumor specimen for each treatment group. MVD was of Mycoplasma using MycoAlert (Cambrex Bio Science) according calculated by viewing 10 representative 200 fields per slide to the manufacturer's instructions. in each treatment group and counting the number of micro- vessels per field. A microvessel was definedasanopenlumen Nab-paclitaxel and topotecan reconstitution with at least one CD31-positive cell immediately adjacent to it Nab-paclitaxel was obtained from Abraxis (Costa Mesa; ref. 48). (49, 50). The apoptotic index for each treatment group was The nab-paclitaxel dosage was determined by the paclitaxel con- quantified as the number of apoptotic tumor cells in 10 tent of the albumin-bound formulation (48). According to the randomly selected 200 high-power fields per slide, excluding manufacturer's instructions, nab-paclitaxel was reconstituted in areas of necrosis. TSP-1 expression was quantified using ImageJ normal saline to a concentration of 5 mg/mL paclitaxel, prepared software. fresh each day, and administered within an hour of mixing. Hypoxic areas on tumor samples were then evaluated using Topotecan was obtained from GlaxoSmithKline (23). According immunohistochemical staining for pimonidazole protein to the manufacturer's instructions, the lyophilized powder was adducts. Hypoxic areas were determined using the Hypoxyprobe reconstituted to 10 mmol/L in Tri-HCl (pH 4.0), then diluted in staining kit (NPI, Inc.). Paraffin sections were used, and antigen sterile water for oral therapy before use. retrieval was performed using a citrate solution in a microwave processor. Sections were probed with Hypoxyprobe Mab-1 Western blot analyses (1:500) overnight at 4C; after washing, slides were incubated HeyA8 cells were treated with nab-paclitaxel, metronomic with secondary antibody (1:500) for 1 hour at room temperature. topotecan or vehicle (normal saline) for 24 to 96 hours. The hypoxic area was measured using ImageJ software.

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AB2 2

SKOV3ip1 HeyA8 1 1 Tumor weight (g) Tumor 0 weight (g) Tumor 0

MTD MTD Vehicle MET 5 Vehicle MET 5 MET 2.5 MET 10 MET 2.5 MET 10 C

1.50 3 2 HeyA8-MDR SKOV3ip1 HeyA8

0.75 1.5 1 Tumor weight (g) Tumor Tumor weight (g) Tumor 0 0 weight (g) Tumor 0

Vehicle Vehicle Vehicle Topotecan Topotecan Topotecan nab-Paclitaxel nab-Paclitaxel nab-Paclitaxel nab-Paclitaxel+Topotecan nab-Paclitaxel+Topotecan nab-Paclitaxel+Topotecan D E WBC (103/mL) Hgb (g/dL) Platelets (103/mL) 100 0 2.5 5 0 7.5 15 0 625 1,250 Combination 80 Vehicle

Topotecan 60 nab-Paclitaxel

40 Topotecan

nab-Paclitaxel 20 nab-Paclitaxel+

Cumulative survival (%) Topotecan P Control 0 < 0.001 0 10 20 30 40 50 60 Time (days)

Figure 1. Tumor growth inhibition with metronomic nab-paclitaxel alone and in combination with metronomic topotecan. Nab-paclitaxel dose-ﬁnding experiment in SKOV3ip1 (A) and HeyA8 (B) tumor models of ovarian carcinoma in athymic mice. SKOV3ip1 or HeyA8 tumor–bearing mice were given nab-paclitaxel at MTD or metronomic (MET) doses of 2.5, 5, or 10 mg/kg. MET doses were administered daily for SKOV3ip1 and on alternating days for HeyA8. Mean tumor weights (SE; n ¼ 5 per group) are shown; , P < 0.05 compared with vehicle. C, SKOV3ip1, HeyA8, and HeyA8-MDR tumor–bearing mice were randomly allocated to four groups and treated with either: vehicle (control), metronomic nab-paclitaxel alone (2.5 mg/kg i.p. every other day), metronomic topotecan alone (0.5 mg/kg orally every day), or metronomic nab-paclitaxel plus metronomic topotecan. Mean tumor weights (SEM) are shown; , P < 0.01; , P < 0.03; , P < 0.05 compared with vehicle. D, survival of mice treated with vehicle (control), metronomic nab-paclitaxel, or metronomic topotecan alone and combination metronomic nab-paclitaxel plus metronomic topotecan. Nude mice (n ¼ 10 per group) were injected with HeyA8 cells and subsequently randomized into the different treatment regimens. Treatment was continued until mice were individually moribund, and the days of life were recorded. E, mean (SE) temporal complete blood count parameters in the SKOV3ip1 model from the therapy experiments at baseline (solid bars) and after 3 weeks of treatment (white bars). WBC, white blood count; Hgb, hemoglobin.

Dose-finding experiments using nab-paclitaxel and topotecan and nodule formation were recorded and analyzed. An additional in orthotopic murine models dose-finding experiment was then performed to ascertain whether We injected SKOV3ip1 tumor cells i.p. (1 106 cells) into greater distinction could be drawn between metronomic treat- female athymic mice. Fourteen days after tumor cell injection, ment groups. We conducted an additional dose-finding experi- mice were randomized into one of five treatment groups (n ¼ 5/ ment using metronomic dosing of nab-paclitaxel at 2.5, 5, and group): vehicle alone, MTD paclitaxel (30 mg/kg daily for 5 10 mg/kg every other day using HeyA8 cells (n ¼ 5/group). consecutive days in a 28-day cycle), 2.5, 5, or 10 mg/kg metro- To evaluate whether therapeutic effects could be augmented nomic (daily) nab-paclitaxel. Nab-paclitaxel was administered by while maintaining treatment tolerability, we administered met- i.p. injection. All mice were collectively sacrificed and tumors were ronomic topotecan by oral gavage daily at dose levels previ- harvested when the control group became moribund. Blood ously identifiedandreportedbyourgroup(i.e.,0.5mg/kg samples were collected by intracardiac withdrawal before necrop- daily; ref. 20). Nab-paclitaxel was administered i.p. at metro- sy for hematologic assessment. Mice were monitored daily for nomic doses every other day as determined by our dose-finding signs of toxic effects and intolerance to therapy. Tumor weights experiments. All treatments were started 7 days after tumor cell

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Figure 2. Biologic effects of metronomic nab-paclitaxel and metronomic topotecan. A, representative images of HeyA8 tumor samples immunohistochemically stained for CD31, cleaved caspase-3, and Ki-67 from mice treated with vehicle, metronomic nab-paclitaxel (2.5 mg/kg i.p. every other day), metronomic topotecan (0.5 mg/kg orally every day), or a combination of metronomic nab-paclitaxel and metronomic topotecan. Error bars, SEM; , P < 0.05 compared with vehicle; , P < 0.01 compared with vehicle. hpf, high-power ﬁeld. (Continued on the following page.)

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inoculation, and mice were randomized into one of four paclitaxel and metronomic topotecan. In the control, MTD nab- treatment groups (n ¼ 10). Mice were monitored daily for paclitaxel, and MTD topotecan wells, the culture medium was adverse effects and drug tolerance. All animals were sacrificed aspirated every 24 hours and replaced with fresh culture medium and tumors were harvested at necropsy when the control mice containing no drug. In the metronomic nab-paclitaxel, metro- began to appear moribund, approximately 3 to 4 weeks after nomic topotecan, and metronomic combination wells, the cul- the initiation of therapy, depending on the cell line used (45). ture medium was aspirated every 24 hours and replaced with a Mouse weight, tumor weight, tumor distribution, and ascites fresh preparation of medium containing treatment at the con- volume were recorded. centrations listed above. After 72 hours, the culture medium from each well was collected. Animals and orthotopic model of ovarian cancer RF-24 cells were maintained in DMEM supplemented with In vivo studies used female athymic mice (NCr-nu) purchased pyruvate, amino acids, and penicillin/streptomycin at 37C. A from the National Cancer Institute-Frederick Cancer Research and 96-well plate was coated with 50 mL of Matrigel, which was Development Center (Frederick, MD). Housing and care in spe- allowed to solidify at 37C for 10 minutes. Next, 20,000 cells cific pathogen-free conditions were provided as stipulated by per well were seeded on the Matrigel and cultured with con- guidelines from the American Association for Accreditation of ditioned media from wells treated with control (no treatment), Laboratory Animal Care, the U.S. Public Health Service Policy on MTD nab-paclitaxel, MTD topotecan, metronomic nab-pacli- Human Care and Use of Laboratory Animals, and the National taxel, metronomic topotecan, or a combination of metronomic Institutes of Health. All studies were approved and monitored by nab-paclitaxel and metronomic topotecan. The cells were incu- The University of Texas MD Anderson Cancer Center Institutional bated at 37C for 16 hours. To assess tube formation, we Animal Care and Use Committee. counted and photographed complete tubes from randomly The development of the orthotopic murine model of advanced chosen fields using an Olympus inverted microscope connected ovarian cancer has been extensively reported by our laboratory to a digital camera. (51). In brief, SKOV3ip1, HeyA8, and HeyA8-MDR cells were collected from 70% to 80% confluent cultures using either 0.25% Statistical analysis Trypsin-EDTA (GIBCO) or 0.1% EDTA, depending on the cell In vivo therapy experiments were powered to detect a 50% line. Cells lifted with trypsin underwent trypsin-neutralization difference in tumor weight (b error ¼ 0.2). The Mann–Whitney with FBS-containing medium before being centrifuged and resus- rank-sum test was used to analyze nonparametric and non-nor- pended in the appropriate volume of serum-free Hank's balanced mally distributed datasets. Survival experiments were analyzed by salt solution (HBSS; Invitrogen) for animal inoculation. Cell lines the Kaplan–Meier method. Statistical analyses were performed lifted with EDTA alone were directly centrifuged at 1,000 rotations using SPSS 12.0 for Windows (SPSS Inc.), and significance was set per minute for 7 minutes at 4 C, washed with PBS, and then at P 0.05 (two-tailed). resuspended in serum-free HBSS at the appropriate concentra- tions for intraperitoneal inoculation: A total of 2.5 105 cells/ 200 mL HBSS for HeyA8 cells or 1 106 cells/200 mL HBSS for Results SKOV3ip1 and HeyA8-MDR cells. Metronomic dosing of nab-paclitaxel Therapy experiments were performed using all three cell lines. To determine the optimal dose and frequency of dosing to Mice were collectively sacrificed when mice in any group appeared effectively inhibit tumor growth, we conducted a dose-finding near moribund, approximately 4 weeks after initiation of therapy, experiment with nab-paclitaxel. Female nude mice (n ¼ 5/group) depending on the cell line. Tumors and ascites were harvested were injected with SKOV3ip1 cells i.p. and were treated with at the time of necropsy from the peritoneal cavities of mice. vehicle, a single dose of MTD nab-paclitaxel (30 mg/kg i.p. daily The number of tumor nodules were quantified, and total tumor for 5 consecutive days in a 28-day cycle) or metronomic nab- weights were determined. Additional tumor tissue for hema- paclitaxel at daily doses of 2.5, 5, or 10 mg/kg. All daily metro- toxylin and eosin staining and immunohistochemistry (see nomic dosing levels of nab-paclitaxel produced profound inhi- above) was fixed in formalin at the time of tumor collection bition of tumor growth (Fig. 1A). and then embedded in paraffin. Paraffin sections were uni- These findings led us to perform additional dose-finding formly cut to 5 mm. experiments using an alternating day dosing frequency in the HeyA8 tumor model. This dosing schedule resulted in less Endothelial cell tube formation assay tumor inhibition (Fig. 1B). Moreover, pancytopenia was HeyA8 cells were seeded at a density of 100,000 per well in a 6- observed in mice that were treated with nab-paclitaxel doses well plate and allowed to attach overnight. The culture medium of 5 and 10 mg/kg after 2 weeks, but not with the 2.5 mg/kg was then aspirated and replaced with fresh culture medium dose (Supplementary Fig. S1). Therefore, we selected 2.5 containing 40 nmol/L MTD nab-paclitaxel, 25 nmol/L MTD mg/kg nab-paclitaxel i.p. every other day as the optimal dose topotecan, 20 nmol/L metronomic nab-paclitaxel, 25 nmol/L for subsequent combination experiments. MTD dosing and metronomic topotecan, or a combination of metronomic nab- metronomic nab-paclitaxel at 10 mg/kg would abrogate tumor

(Continued.) B, representative images of HeyA8-MDR samples immunohistochemically stained for CD31, cleaved caspase-3, and Ki-67 from mice treated with vehicle, metronomic nab-paclitaxel (2.5 mg/kg i.p. every other day), metronomic topotecan (0.5 mg/kg orally every day), or a combination of metronomic nab-paclitaxel and metronomic topotecan. (original magniﬁcation 100; scale bar, 100 mm). Error bars, SEM; , P < 0.05 compared with vehicle; , P < 0.01 compared with vehicle. hpf, high-power ﬁeld. C, the percentage of tissue showing hypoxia in SKOV3ip1 tumor cells from each treatment group. Error bars, SE; , P < 0.05 compared with vehicle. D, Western blot images depicting the effect of vehicle, metronomic topotecan, maximally tolerated dose (MTD) nab-paclitaxel, and combination on carbonic anhydrase IX (CAIX) expression from tumors in the HeyA8 orthotopic ovarian cancer model.

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growth and prevent assessment of dual metronomic therapy. topotecan resulted in a 64% reduction of MVD (P < 0.01) We had previously selected the dosing schedule for metro- compared with vehicle (Fig. 2A). In the HeyA8-MDR model, nomic topotecan (20). treatment with metronomic nab-paclitaxel or topotecan had no effect on MVD; however, treatment with the combination of In vivo effects of metronomic nab-paclitaxel and topotecan on metronomic nab-paclitaxel and metronomic topotecan resulted ovarian carcinoma in a nearly 60% reduction of MVD compared with vehicle The next experiment had 4 treatment groups: (i) vehicle, (P < 0.01; Fig. 2B). (ii) metronomic nab-paclitaxel (2.5 mg/kg i.p. every other day), To determine whether apoptosis contributed to the antitu- (iii) metronomic topotecan (0.5 mg/kg daily via oral gavage), and mor effects observed with metronomic nab-paclitaxel and (iv) metronomic nab-paclitaxel every other day plus metronomic metronomic topotecan, we conducted immunohistochemical topotecan daily. Compared with vehicle, treatment with metro- staining for cleaved caspase-3 in tumors harvested after the nomic nab-paclitaxel alone resulted in a 62% reduction in tumor therapy experiments. HeyA8 tumors from animals treated weight (P < 0.01), and treatment with the combination of met- with metronomic nab-paclitaxel or topotecan alone had a ronomic nab-paclitaxel and metronomic topotecan resulted in a significant increase in apoptotic cells (2.3- and 2.68-fold, 96% reduction in tumor weight (P < 0.01) in the SKOV3ip1 tumor respectively; P < 0.01). Meanwhile, the combination treatment model (Fig. 1C). Similar results were obtained in the HeyA8 had the most profound effect on apoptosis (4.5-fold; P < 0.01; tumor model: Metronomic nab-paclitaxel alone and metronomic Fig. 2A). Furthermore, HeyA8-MDR tumors from mice treated nab-paclitaxel combined with metronomic topotecan elicited with metronomic nab-paclitaxel or topotecan alone resulted >90% reductions in tumor weight (P < 0.03) compared with in a 1.98- and 2.14-fold increase in apoptotic cells, whereas vehicle (Fig. 1C). the combination therapy induced a 4.96-fold increase (Fig. 2B; Recognizing the high prevalence of taxane resistance in recur- P < 0.01). rent ovarian cancer, we also tested the therapeutic potential of We examined treatment effects on tumor cell proliferation by metronomically dosed nab-paclitaxel and topotecan in the tax- calculating the proliferative index after immunohistochemical ane-resistant HeyA8-MDR tumor model. Although metronomic staining for Ki-67 in tumors collected at necropsy at the end of monotherapy with either cytotoxic agent alone did not overcome the therapy experiments. The proliferative index was significantly taxane resistance, the combination of metronomic nab-paclitaxel reduced in mice with HeyA8 tumors treated with either metro- and metronomic topotecan decreased tumor burden by 60% nomic nab-paclitaxel (51%, P < 0.05) or metronomic topotecan (P < 0.05) compared with vehicle, thereby supporting the use of alone (53%, P < 0.05; Fig. 2A). The combination treatment a combination of two metronomic cytotoxic agents to augment resulted in a 75% reduction (P < 0.01) in proliferative index. The the observed therapeutic effect (Fig. 1C). proliferative index in HeyA8-MDR tumors treated with nab-pac- On the basis of these results with regard to inhibition of in vivo litaxel or topotecan was reduced by 39% and 33%, respectively, tumor growth using metronomic regimens, we next examined the whereas the combination treatment significantly reduced prolif- effects of these regimens on survival using the HeyA8 model. eration by 88% (P < 0.01; Fig. 2B). Treatment with metronomic nab-paclitaxel or metronomic topo- To further evaluate the contribution of metronomic therapy on tecan prolonged survival; the most significant effect on survival angiogenesis inhibition and hypoxia, we treated 3 mice from each time was in the combination arm with metronomic nab-paclitaxel treatment arm in the SKOV3ip1 tumor model with a hypoxia and metronomic topotecan, where survival was significantly marker, pimonidazole, before sacrificing them and stained the increased (P < 0.001; Fig. 1D). harvested tumor tissue for pimonidazole protein adducts, which No differences in body weight were identified among treatment represent areas of hypoxia within the solid tumor. Immunohis- groups in any of the models. To assess hematologic tolerance of tochemical staining with pimonidazole showed that treatment the combination therapy, complete blood counts were performed with metronomic nab-paclitaxel alone and metronomic topote- using blood drawn via tail vein phlebotomy just before initiating can alone resulted in hypoxic areas in approximately 10% of the treatment and again after 3 weeks of treatment. In the complete tumor tissue, compared with only 5% after treatment with vehicle blood count analysis, white blood cell and platelet counts did (Fig. 2C). Treatment with the combination of metronomic nab- not differ over the course of treatment in animals treated with paclitaxel and metronomic topotecan resulted in nearly double metronomic nab-paclitaxel alone or in those treated with the percentage of tissue showing hypoxia (19%, P ¼ 0.04; Fig. 2C), metronomic nab-paclitaxel combined with metronomic topo- further supporting the observation that metronomic nab-pacli- tecan (Fig. 1E). Direct observation of the animals' eating habits taxel and metronomic topotecan exert antiangiogenic effects and daily veterinary care of other physical characteristics directly on tumors. Treatment with metronomic topotecan, revealed no obvious deviation from typical bowel or feeding nab-paclitaxel, or combination therapy led to an increase in habits, posture, or mobility among the four treatment groups in carbonic anhydrase IX expression tumors from mice inoculated any of the three models. with HeyA8 cells (Fig. 2D).

Biologic effects of metronomic nab-paclitaxel and metronomic In vitro effects of metronomic nab-paclitaxel compared with topotecan on proliferation, angiogenesis, and apoptosis MTD nab-paclitaxel Given the proposed role of nab-paclitaxel on angiogenesis One possible mechanism underlying the antitumor effects of inhibition, we calculated the MVD of tumors harvested at the metronomic chemotherapy is increased tumor cell expression conclusion of the therapy experiments. In the HeyA8 tumor of antiangiogenic factors such as TSP-1 in response to treat- model, treatment with metronomic nab-paclitaxel or topotecan ment. To test whether metronomic nab-paclitaxel induces decreased MVD by 45% and 41%, respectively (P < 0.05), whereas TSP-1 expression in ovarian cancer cells, we treated HeyA8 the combination of metronomic nab-paclitaxel and metronomic cells with a sub-IC50 dose of nab-paclitaxel (0.5 nmol/L) for

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Figure 3. Effect of metronomic nab-paclitaxel and topotecan on TSP-1 expression. A, Western blot images depicting the effect of control (normal saline) and metronomic nab-paclitaxel on TSP-1 protein expression. HeyA8 cells were continuously treated with either vehicle or 0.5 nmol/L nab-paclitaxel for up to 96 hoursand cell lysates were collected at 24-hour time points. The graph shows the band intensity of TSP-1 normalized to the loading control, vinculin. B, Western blot analysis depicting the effect of control (normal saline), metronomic topotecan (25 nmol/L daily), and the maximally tolerated dose (MTD) of nab-paclitaxel (20 nmol/L once) on TSP-1 expression. Cell lysates were collected after 72 hours. C, tube formation of RF-24 cells after treatment with conditioned media of each treatment group, representative images shown. (original magnification 100; scale bar, 100 mm). D, representative images of HeyA8 tumor samples immunohistochemically stained and quantified for TSP-1 (original magnification 100; scale bar, 100 mm). Error bars, SEM; , P < 0.05 compared with vehicle; , P < 0.01 compared with vehicle. up to 96 hours. In Western blot analysis, we found that protracted metronomic topotecan and MTD nab-paclitaxel could induce exposure of HeyA8 tumor cells to nab-paclitaxel resulted in a TSP-1, we treated HeyA8 cells with nab-paclitaxel at the beginning substantial and time-dependent induction of TSP-1 protein of the experiment and daily topotecan for 72 hours. Western blot expression (Fig. 3A). TSP-1 expression was maximally induced analysis shows that both treatments induced TSP-1 expression after 72 hours of treatment, corresponding to a 2.5 times increase (Fig. 3B) treatment with daily topotecan, MTD nab-paclitaxel and in TSP-1 expression compared with vehicle. To determine whether combination treatments.

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Next, we performed tube formation assays using RF-24 cells shown to reduce normal tissue toxicity and minimize off-treat- exposed to conditioned media from HeyA8 cells that were ment exposure (57). Several clinical trials have shown that more exposed to nab-paclitaxel or topotecan. Tube formation was frequent treatment with lower doses of chemotherapy leads to inhibited in RF-24 endothelial cells exposed to conditioned fewer toxic effects and improved pathologic response rates in media from HeyA8 cells exposed to metronomic nab-paclitaxel patients with breast cancer (3, 57). However, a single metronomic and topotecan whereas the combination treatment had the most regimen is unlikely to have universal efficacy, and novel combi- robust effect (Fig. 3C). nation regimens of metronomic chemotherapy are needed. To investigate whether metronomic dosing increased TSP-1 Our study provides evidence of potent antitumor activity in expression in vivo, we performed immunohistochemistry analyses both taxane-sensitive and taxane-resistant orthotopic models of on HeyA8 tumor samples. Our data show that metronomic nab- metastatic ovarian carcinoma after treatment with metronomic paclitaxel and topotecan increased TSP-1 expression whereas this nab-paclitaxel alone and in combination with metronomic topo- effect was exacerbated in the combination group (Fig. 3D). tecan. With potent antitumor effects in the absence of overt toxic effects that occur with traditional chemotherapy, metronomic Discussion nab-paclitaxel alone or in combination with topotecan is a strategy worthy of further clinical investigation. In this study, we demonstrated marked antitumor activity in ovarian carcinoma models using combination metronomic che- fl motherapy. These effects were mediated, in part, through a Disclosure of Potential Con icts of Interest fl significant reduction in tumor cell proliferation and increased No potential con icts of interest were disclosed. apoptosis. We also demonstrated that metronomic administration of nab-paclitaxel increased levels of TSP-1, thereby disrupting Authors' Contributions angiogenesis and providing a mechanism by which this com- Conception and design: Y.G. Lin, H.J. Dalton, A.K. Sood pound elicits its therapeutic effects. These antitumor and anti- Development of methodology: Y.G. Lin, A.K. Sood Acquisition of data (provided animals, acquired and managed patients, angiogenic effects were validated in both taxane-sensitive and provided facilities, etc.): R.A. Previs, G.N. Armaiz-Pena, Y.G. Lin, A.N. Davis, taxane-resistant tumor models. S. Pradeep, H.J. Dalton, J.M. Hansen, W.M. Merritt, A.K. Sood Our results suggest that, although apoptosis plays a role in Analysis and interpretation of data (e.g., statistical analysis, biostatistics, eliciting the observed antitumor effects of treatment with metro- computational analysis): Y.G. Lin, A.N. Davis, H.J. Dalton, J.M. Hansen, nomic topotecan, the reduction in tumor growth seen with R. Coleman, A.K. Sood metronomic nab-paclitaxel appears to be more attributable to Writing, review, and/or revision of the manuscript: R.A. Previs, G.N. Armaiz- Pena, Y.G. Lin, A.N. Davis, H.J. Dalton, J.M. Hansen, A.M. Nick, R. Coleman, the inhibition of angiogenic factors, resulting in large areas of A.K. Sood hypoxia within the tumor. In addition, a resultant increase in Administrative, technical, or material support (i.e., reporting or organizing CAIX expression was observed in vitro treatment of cells with data, constructing databases): Y.G. Lin, R.R. Langley, R. Coleman, A.K. Sood metronomic topotecan and nab-paclitaxel. The profound inhibi- Study supervision: A.K. Sood tion of endothelial tube formation by conditioned media from cells treated with metronomic nab-paclitaxel alone and in com- Acknowledgments bination with metronomic topotecan supports existing evidence The authors thank Nicholas Jennings, Donna Reynolds and Dr. Yun-Fang that metronomic dosing not only has direct cytotoxic effects on Wang for their insightful discussions and expertise. endothelial cells, but also indirectly inhibits angiogenesis through the release of mediators from the cancer cell into the tumor Grant Support microenvironment (52). Financial support was provided by the NIH (P50 CA098258, CA 109298, Angiogenic pathways and the tumor microvasculature are P50 CA083639, U54 CA151668, and MD Anderson's Cancer Center Support attractive targets for cancer treatment, as supported by the robust grant CA016672), the Ovarian Cancer Research Fund, Inc. (Program Project Development Grant), the U.S. Department of Defense (OC073399, OC093146, number of ongoing clinical trials in this arena (6). Nevertheless, and BC085265), the Ann Rife Cox Chair in Gynecology, the Betty Anne both intrinsic and acquired resistance to antiangiogenic drugs are Asche Murray Distinguished Professorship, the RGK Foundation, and the emerging as clinically relevant issues (53, 54). Mounting data Gilder Foundation (to A.K. Sood). R.A. Previs, J.M. Hansen, and H.J. Dalton suggest that administering traditional cytotoxic agents at metro- were supported by an NCI-DHHS-NIH T32 training grant (T32 CA101642). nomic doses exerts antiangiogenic effects (6, 52, 55, 56). Metro- G.N. Armaiz-Pena was partially supported by the NIH U54CA163071 and nomic paclitaxel has stronger antitumor activity in terms of G12MD007579 grants. The costs of publication of this article were defrayed in part by the payment of suppressing primary and metastatic breast tumors, with fewer page charges. This article must therefore be hereby marked advertisement in side effects and stronger antiangiogenic and anti-lymphangio- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. genic activities, than MTD therapy. Moreover, metronomic paclitaxel chemotherapy causes a substantial increase in the expres- Received August 13, 2014; revised August 24, 2015; accepted September 15, sion of TSP-1. Metronomic dosing of chemotherapeutics has been 2015; published OnlineFirst October 29, 2015.

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Dual Metronomic Chemotherapy with Nab-Paclitaxel and Topotecan Has Potent Antiangiogenic Activity in Ovarian Cancer

Rebecca A. Previs, Guillermo N. Armaiz-Pena, Yvonne G. Lin, et al.

Mol Cancer Ther Published OnlineFirst October 29, 2015.

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